The presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) dictates 5-HT inactivation following release in the brain and periphery and is a major target for antidepressant medications as well as psychostimulants, including cocaine and 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy). Studies over the past two decades by our group have progressively illuminated the structural basis for 5-HT clearance and the mechanisms resident within serotonergic neurons and terminals that provide for control of membrane SERT activity, including evidence of trafficking-dependent and independent pathways. Furthermore, we have identified multiple, naturally occurring SERT coding variants (e.g. Gly56Ala) that disrupt SERT regulation in vitro. In the prior iteration of this project, we identified protein kinase G subtype 1 (PKG1) and p38 MAPKa as critical determinants of receptor-driven SERT trafficking and catalytic activation, respectively. In this renewal application, we propose to advance these studies through an integrated series of in vitro, ex vivo and in vivo analyses capitalizing on our increased understanding of receptor systems activating these pathways and the development of novel transgenic mouse models. Through this effort we seek to not only explore the fundamental biology underlying SERT regulation, but also to provide proof of concept for novel therapeutic approaches to treat 5-HT associated brain disorders. In Specific Aim I, we seek to establish the structural basis of SERT regulation by A3A adenosine receptors (A3ARs) and IL-1 receptors (IL-1Rs) that provide for rapid SERT modulation. Specifically, we investigate the hypothesis that these receptors participate in, and signal through physical complexes with SERT and rely on cGMP-dependent SERT phosphorylation as well as altred membrane compartmentation. Finally, we pursue domains and residues that establish the differential control of SERT imparted by these signaling pathways. In Specific Aim II, we elucidate the impact in vitro of naturaly- occuring, human SERT protein variation on receptor- and kinase-mediated SERT regulation and extend our efforts to ask whether coding variation in A3ARs and IL-1Rs mimics the effects of SERT coding variation. In Specific Aim III, we seek to translate our understanding of A3AR, IL-1R and p38 MAPKa pathways to examine the impact on 5-HT homeostasis, 5-HT clearance and synaptic 5-HT signaling in brain preparations. This Aim will make use of both pharmacological approaches as well as a novel line displaying adult, 5-HT-neuron- specific elimination of p38 MAPKa. In Specific Aim IV, we.examine a novel line of mice engineered to expess a disease-associated, hyperphosphorylated coding variant of SERT (SERT Gly56Ala) for effects on phosphorylation, protein complexes, 5-HT clearance and synaptic 5-HT signaling in native preparations. Finally, these mice are explored for impact on behavioral effects of the psychostimulants cocaine and MDMA.